title: Inhibition of four-and-a-half LIM domain protein-2 increases survival, migratory capacity, and paracrine function of human early outgrowth cells through activation of the sphingosine kinase-1 pathway: implications for endothelial regeneration.
abstract: BACKGROUND
  Inhibition of four-and-a-half LIM domain protein-2 (FHL2) attenuates atherosclerotic lesion formation and increases endothelial cell migration. Early outgrowth cells (EOCs) contribute substantially to endothelial repair.
  
  
  OBJECTIVE
  We investigated the role of FHL2 in the regulation of EOCs.
  
  
  RESULTS
  Human EOCs were cultured from peripheral blood. FHL2 knockdown in EOCs by siRNA resulted in increased EOC numbers and reduced apoptosis, as indicated by decreased cleaved caspase-III and reduced Bax/Bcl-2 expression ratio. This was mediated through increased phosphorylation and membrane translocation of sphingosine kinase-1, increased sphingosine-1-phosphate levels, and Akt phosphorylation. FHL2 knockdown increased stromal cell-derived factor-1-induced EOC migration through upregulation of αv/β3, αv/β5, and β2 integrins, associated with increased cortactin expression. Reduced apoptosis, increased EOC migration, and cortactin upregulation by FHL2 siRNA were prevented by CAY10621, the sphingosine kinase-1 inhibitor, and the sphingosine-1-phosphate receptor-1/-3 antagonist VPC23019. These findings were confirmed using spleen-derived EOCs from FHL2(-/-) mice. Apoptosis was decreased and migration increased in endothelial cells exposed to the conditioned medium of FHL2(-/-) versus wild-type (WT) EOCs. These paracrine effects were abolished by VPC23019. Importantly, reendothelialization after focal carotid endothelial injury in WT mice was significantly increased after intravenous injection of FHL2(-/-) versus WT EOCs.
  
  
  CONCLUSIONS
  Our findings suggest that FHL2 negatively regulates EOC survival, migration, and paracrine function. FHL2 inhibition in EOCs reduces apoptosis and enhances survival and migratory capacity of both EOCs and surrounding endothelial cells by activation of the sphingosine kinase-1/sphingosine-1-phosphate pathway, resulting in improvement of endothelial regeneration.
authors.affiliation: From Lady Davis Institute for Medical Research, Jewish General Hospital, Department of Medicine, McGill University, Montréal, Québec, Canada.
authors.forename: Talin
authors.initials: T
authors.lastname: Ebrahimian
authors.forename: Omar
authors.initials: O
authors.lastname: Arfa
authors.forename: Stefania
authors.initials: S
authors.lastname: Simeone
authors.forename: Catherine A
authors.initials: CA
authors.lastname: Lemarié
authors.forename: Stéphanie
authors.initials: S
authors.lastname: Lehoux
authors.forename: Sven
authors.initials: S
authors.lastname: Wassmann
mesh_terms: D000328:Adult; D000818:Animals; D017209:Apoptosis; D051017:Apoptosis Regulatory Proteins / Q000378:metabolism; D002339:Carotid Arteries / Q000166:cytology; D002465:Cell Movement; D002470:Cell Survival; D002478:Cells, Cultured; D051356:Cortactin / Q000235:genetics / Q000378:metabolism; D017077:Culture Media, Conditioned / Q000494:pharmacology; D042783:Endothelial Cells / Q000187:drug effects / Q000378:metabolism* / Q000502:physiology; D004730:Endothelium, Vascular / Q000166:cytology / Q000293:injuries / Q000378:metabolism; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005260:Female; D006801:Humans; D016023:Integrins / Q000235:genetics / Q000378:metabolism; D060850:LIM-Homeodomain Proteins / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D007963:Leukocytes, Mononuclear / Q000378:metabolism / Q000502:physiology; D008297:Male; D051379:Mice; D008810:Mice, Inbred C57BL; D009124:Muscle Proteins / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D019899:Paracrine Communication; D017853:Phosphotransferases (Alcohol Group Acceptor) / Q000037:antagonists & inhibitors / Q000378:metabolism*; D049349:Receptors, Lysosphingolipid / Q000037:antagonists & inhibitors / Q000378:metabolism; D013154:Spleen / Q000166:cytology; D014157:Transcription Factors / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D014945:Wound Healing; D000099157:Sphingosine Kinase
chemical_list: D051017:Apoptosis Regulatory Proteins; D051356:Cortactin; D017077:Culture Media, Conditioned; D004791:Enzyme Inhibitors; D016023:Integrins; D060850:LIM-Homeodomain Proteins; D009124:Muscle Proteins; D017853:Phosphotransferases (Alcohol Group Acceptor); D049349:Receptors, Lysosphingolipid; D014157:Transcription Factors; D000099157:Sphingosine Kinase; C105987:FHL2 protein, human; C491382:Fhl2 protein, mouse
keywords: apoptosis; endothelium; physiology; signal transduction; stem cells